The invention concerns gelatin membrane filters for the collection of microorganisms from gases and a process for making such filters.
The gelatin membranes of the invention find utility in the collection of microorganisms from gaseous media such as air, and are particularly suitable for use in the fields of pharmaceuticals, biotech research and development, food and beverage industries, environmental protection, waste management and in equipment for the determination of bacterial counts. In combination with an air microorganism collection device, such membranes permit collection of bacteria, viruses, yeasts and fungi, in order to determine their concentration in a given space. Such monitoring is the first step for the timely introduction of measures to protect persons and products from harm due to an unduly high concentration of microorganisms in, for example, the air of a room.
In special rooms with unusual requirements as to the state of the air, such as in air-conditioned rooms, clean rooms, and intensive care units, the air is regularly checked for its microorganism count. Since, as a general rule, it is filtered air that is tested which by its nature exhibits a low microorganism count, normally large volumes must be tested in order to collect sufficient microorganisms for a reliable report. Air samples are filtered through an appropriately chosen filter such as sterile membrane filters with pore sizes in the microfiltration range. Typically, such filters are of cellulose nitrate, cellulose acetate and gelatin. See, for example, DE 11 73 640. Especially well adapted to the task are gelatin membrane filters, which are generally thought to remain sufficiently moist to be conducive to propagation of retained microorganisms.
Following sampling the microorganism-laden gelatin membrane filters are either placed on an agar growth medium to incubate or stored in a sterile solution such as peptone water or an isotonic saline solution to permit aliquot portions to be incubated later. In the case of deposit on an agar growth medium, out of the collected individual bacterial aggregates, colonies of microorganisms grow while the gelatin membrane filter dissolves and disappears, thus permitting a microorganism count directly on the agar.
When nutrients are supplied to a gelatin membrane filter for growing microorganisms, it has been suggested to add buffering materials, coloring substances chemicals capable of absorbing biologically poisonous materials and/or counteracting traces of heavy metals or even damaging gases contained in the original air sample. See DE 11 73 640 The disadvantage of this is that in spite of the ready supply of nutrients, apparently due to the added substances, substantially fewer detectable colonies propagate from the microorganisms collected on the membrane than were originally present in the contaminated medium, giving a false or inaccurate count.
Thus, a principal aspect of the present invention is the provision of a gelatin membrane filter for the collection of microorganisms from gases, wherein the number of detectable viable microorganisms is substantially increased relative to the number obtainable with known gelatin membrane filters. A closely related aspect is the provision of a process for the manufacture of such a gelatin membrane filter.
Surprisingly, it has been discovered that the number of detectable microorganisms collected on gelatin membrane filters is substantially increased, thereby substantially enhancing bacterial count accuracy, when the gelatin membrane filter contains so-called xe2x80x9cosmoprotectivexe2x80x9d agents such as inositol, betaine, lysine and oxyneurine. Trimethylammonioacetate (TMAA) has been found to be a particularly effective osmoprotective agent insofar as achieving the enhanced and more accurate bacterial count object of the invention.
Contrary to prevailing thought, upon collection by a gelatin membrane filter, a portion of the microorganisms loses so much of its cellular water content that the microorganisms therein are no longer viable, meaning that, upon incubation, no colonies from such a portion can be detected. This phenomenon was discovered from the fact that about half of a known bacteria count for a given sample could be detected when the bacteria-capturing gelatin membrane filter was subjected to nine minutes of air flow instead of the usual one minute. This was not expected, because conventional wisdom was that microorganisms collected on gelatin membrane filters remain moist and able to propagate. See, for example, xe2x80x9cLaboratory Filtration, Microbiology, Electrophoresisxe2x80x9d page 14, Sartorius GmbH (1984).
In a preferred embodiment of the invention, the gelatin membrane filters contain at least one osmoprotective agent in sufficient amount that the number of viable microorganisms is doubled as compared to gelatin membrane filters not containing such an osmoprotective agent.
The discovery has further been made that the inclusion of an osmoprotective agent in the gelatin membrane filters, when such filters are exposed to a variable through-flow of gases containing a predetermined number of microorganisms, leads to varying portions of viable microorganisms. Trimethylammonioacetate (TMAA) has been found to be a particularly effective osmoprotective agent in the present invention, especially when it is present in the gelatin membrane casting solution in amounts of between 0.005 and 0.75 wt % relative to the gelatin content. The casting solution preferably comprises an aqueous, homogenous solution containing 4.6 to 5.6 wt % gelatin and 38 to 46 wt % ethanol. This makeup is advantageous for promoting stability of the membrane, while the gelatin itself may be stabilized by a binder such as polyvinyl alcohol (PVA) or starch.
Because of the structure of gelatin, it is preferable that the osmoprotective agent is distributed as uniformly as possible throughout the gelatin matrix. If the osmoprotective agent(s) are distributed only on the outer surface of the gelatin membrane matrix, there is a risk that, after lengthy storage, a portion of the osmoprotective agent(s) in the inner matrix migrates away, leading to possible total ineffectiveness. For this reason, the osmoprotective agent(s) are preferably incorporated into the membrane matrix homogenously by means of a phase inversion casting process, whereby the osmoprotective agent(s) are incorporated directly into the membrane casting solution.
Alternatively, the osmoprotective agent(s) can be introduced into the membrane matrix in a fixing or precipitation bath where precipitation of the gelatin membrane filter takes place. Such a precipitation bath preferably comprises methyl acetate containing 10 to 20 wt % of an alcohol, preferably methanol, and up to 2 wt % of the osmoprotective agent(s). At the time of its initial introduction to the precipitation bath, the gelatin membrane is in a partially gelled phase and not yet completely formed, so that the osmoprotective agent(s) can permeate the entire matrix of the membrane.
The gelatin membrane filters of the present invention exhibit a high degree of mechanical stability, as compared to known gelatin membrane filters, such as those disclosed in DE 11 73 640 Because of this advantage, the ability to manipulate such membranes is greatly improved, and at the same time they have an improved air flux by a factor of at least two. Because of this, the subject membranes are particularly well suited for large air through-put for the collection of bacteria, permitting reduction of the amount of time for the collection of microorganisms, and further permitting the testing of gases having an extremely low loading of microorganisms, since greater volumes can be filtered without impairing or destroying the effectiveness of the gelatin membrane filters.
A preferred process for the manufacture of the gelatin membrane filter of the present invention comprises the following steps:
(a) providing an aqueous, substantially homogenous membrane casting solution comprising gelatin, ethanol and at least one osmoprotective agent;
(b) applying a thin film of the casting solution of step (a) to a substrate;
(c) allowing the thin film from step (b) to gel to form a partially gelled membrane;
(d) immersing the partially gelled membrane from step (c) in a precipitation bath to form a gelatin membrane filter; and
(e) drying the gelatin membrane filter from step (d).
Alternatively, the osmoprotective agent may be included in the precipitation bath in step (d) instead of in step (a).
In a preferred embodiment of the process, the casting solution contains between 0.005 and 0.75 wt % TMAA relative to the amount of gelatin. In a further embodiment of the invention, in order to increase the mechanical stability of the gelatin membrane filter, 0.02 to 0.1 wt % of a binder such as PVA or starch may be added to the casting solution relative to the total weight of all components of the membrane casting solution.
In a yet another embodiment of the invention, two precipitation baths are used in step (d).
Specifically, a first bath of methyl acetate with 10 to 20 wt % of an alcohol, preferably methanol, is used to initially gel the membrane, where it remains for up to three hours at room temperature. It is then transferred to a second bath of pure methyl acetate for up to three hours at room temperature. Drying, then sterilization, preferably by gamma rays, follow.